Previous manual mixing studies demonstrating decreased Ca2+ uptake activity in cardiac muscle sarcoplasmic reticulum (SR) with age have been extended to include measurements of transport function carried out on a physiologic (milli-second) time scale. The results demonstrate linear rates of ATP-dependent transport activity that are ten times faster than those previously determined by manual filtration (time scale of seconds to minutes). Active Ca2+ accumulation in SR isolated from old rat myocardium showed an average decline in activity of 43% compared to that found in SR prepared from young adult rat hearts (p Greater than .05). These findings lend additional support to the hypothesis relating the prolonged relaxation time in senescent myocardium to a decline in SR Ca2+ pump activity. The kinetics of the phosphoenzyme transition E1P Greater than E2P in the Na,K-ATPase reaction cycle were investigated. The results indicate that the transition is among the fastest reactions of the catalytic cycle and are consistent with a model in which de-occlusion of the transported ions are rate-limiting in the translocation sequence.